Our laboratory has been studying the mechanism of action of the 70 kDa class of heat shock proteins (Hsp70s), which have been termed molecular chaperones because they are involved in the folding and unfolding of proteins and in the formation and dissociation of protein complexes. In these studies we have concentrated on exploring the role of Hsp70 in endocytosis, in particular its ability to uncoat clathrin-coated vesicles. In many of their activities the Hsp70s require cofactors known as J-domain proteins that induce protein substrates to bind to Hsp70, and we previously discovered that uncoating also requires a J-domain protein, the 100 kDa clathrin assembly protein (AP), auxilin. Auxilin is a nerve specific protein and we later discovered that the non-neuronal homolog of auxilin is the 150 kDa protein GAK. During the previous year we showed that C. elegans has a single gene for auxilin and when auxilin expression is inhibited by RNA-mediated interference, there is a marked inhibition of clathrin-mediated endocytosis which in turn causes the worms to arrest during larval development. We also showed that yeast has a single gene for auxilin and that when this gene is deleted the resulting haploid yeast mutants showed an increase in clathrin-coated vesicles and a corresponding decrease in free clathrin in the cytosol. In addition, there was a marked decrease in transport of both carboxypeptidase Y and the G-protein-coupled receptor Ste3 to the vacuole; transport of both of these proteins normally occurs through clathrin-mediated endocytosis. From these data, we concluded that uncoating of clathrin-coated vesicles by Hsp70 and an auxilin homolog is a fundamental step in clathrin-mediated endocytosis. We are now well on the way to obtaining both auxilin and GAK knock-out mice to determine the function of these genes in mammals. During the previous year we also began an investigation of whether dissociation and rebinding of clathrin, i.e. clathrin exchange, is a normal part of clathrin-mediated endocytosis independent of the irreversible dissociation of clathrin that occurs after vesiculation takes place, and also whether Hsc70 and auxilin are involved in this exchange. By blocking clathrin-mediated endocytosis under conditions where clathrin-coated pits on the plasma membrane remain intact, we demonstrated that, in the presence of ATP, clathrin in clathrin-coated pits exchanges with free clathrin in the cytosol. These data suggested that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits and therefore may be involved in the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. These data also suggested that clathrin-coated pits are dynamic structures and therefore raised the possibility that the clathrin assembly protein AP2 in clathrin-coated pits might also exchange with free AP2 in the cytosol. Therefore, during the past year we investigated this question and also investigated whether clathrin and AP1 bound to the trans-Golgi network exchange with free clathrin and AP1 in the cytosol, respectively. As we observed for clathrin, when clathrin-mediated endocytosis was blocked under conditions where clathrin-coated pits remain intact, AP2 in the clathrin-coated pits exchanged with free AP2 and this exchange occurred at about the same rate as clathrin exchange. Similarly, we found that at low temperature where transport out of the trans-Golgi network is blocked, both clathrin and AP1 exchanged with free clathrin and AP1 in the cytosol, respectively. We also previously found that, when clathrin-mediated endocytosis at the plasma membrane was blocked by hypertonic sucrose or K depletion, conditions that have been reported to affect the structure of clathrin-coated pits, clathrin exchange was completely blocked. Therefore, in the present study we investigated how hypertonic sucrose and K depletion affected AP2 exchange at the plasma membrane and clathrin and AP1 exchange at the trans-Golgi network. Interestingly, we found that both hypertonic sucrose and K depletion not only blocked clathrin exchange at the plamsa membrane but also at the trans-Golgi network. However, both AP2 at the plasma membrane and AP1 at the trans-Golgi network continued to exchange under these conditions. We conclude that clathrin-coated pits at both the plasma membrane and the trans-Golgi network are dynamic structures that show rapid exchange of both clathrin and APs. In addition, we conclude that, although clathrin and APs generally exhange at about the same rate, APs are able to exchange independently of clathrin when clathrin exchange is blocked.